The present invention relates to a phosphinic peptide derivative, capable of selectively inhibiting the N-terminal site of human angiotension conversion enzyme (ACE) without the second active site of ACE.
Said peptide derivatives which are selective inhibitors of the N-terminal site of ACE may be used for therapeutic purposes to protest haematopoietic strain cells of patients undergoing aggressive chemotherapy or radiotherapy.
In the 1980s, the development of pseudo-peptide ACE inhibitors genuinely revolutionised the treatment of arterial hypertension, heart failure and chronic kidney failure. In this way, there is now a wide range of synthetic ACE inhibitors which are used in clinical practice. Of these, captopril, enalapril and fosinopril, the formulas of which are given in FIG. 1, are known.
In parallel with this work, due to the cloning of ACE enzyme in 1988, P. Corvol""s group was able to demonstrate the existence of two active sites in said enzyme, as described by Soubrier et al. in Proc. Natl. Acad. Sci. USA (1988) 85, 9386-9390 [1].
The formal demonstration that said two active ACE site control separate physiological functions in humans, represents another revolution in this field and has significant consequences in therapeutic terms. Given that all the ACE inhibitors developed to date inhibit in vitro both active ACE sites to a similar degree, the physiological function of said two active sites in vivo cannot be treated with this type of compound. However, the first inhibitors capable of discriminating between the two active ACE sites would be extremely valuable.
ACE hydrolyses several natural substrates involved in regulating arterial pressure, the circulating blood volume and cardiovascular haemodynamics. The main substrate is angiotensin I, an inactive decapeptide, which is activated to angiotension II, a vasopressor and antinatriuretic peptide, after hydrolysis of the His-Leu carboxyterminal dipeptide. In parallel, ACE inactivates bradykinin, a vasodilator and natriuretic peptide, into a heptapeptide and then into a pentapeptide, both inactive. The two N- and C-terminal sites of ACE are involved in this hydrolysis in a similar fashion.
A specific function of the N-terminal of ACE was recently identified by P. Corvol""s group. As described by Lenfant et al. in Proc. Natl. Acad. Sci. USA (1989), 86, 779-782 [2], the N-Acetyl-Seryl-Aspartyl-Lysyl-Proline (AcSDKP) is a natural circulating inhibitor of the entry into S phase of haematopoietic strain cells. It also inhibits the entry into S phase of other cell types such as hepatocytes in regeneration phase, lymphocytes and several stable cell lines, as described by Lombard et al, in Cell. Tissue. Kinet (1990) 23, 99-103 [3]. The inhibitory action of AcSDKP on the cell cycle of haematopoietic cells is specific to normal haematopoietic cells; leucaemic cells are not concerned. Therefore, AcSDKp was proposed as a therapeutic agent for the protection of medullary progenitors during chemotherapy. In fact, administering AcSDKP prolongs the survival of mice treated with cytotoxic agents (Bodgen et al, Ann. N.Y. Acad. Sci. (1991) 628, 126-139 [4]). As described by Rousseau et al in J. Biol. Chem. (1995) 270, 3656-3661 [5] and Azizi et al in J. Clin. Invest. (1996) 97, 839-844 [6], AcSDKP is hydrolysed in vitro and in vivo by ACE, and more particularly by the N-terminal domain of the enzyme. In vitro, AcSDKP is hydrolysed 50 times more quickly by the N-terminal domain than by the C-terminal domain. This discovery demonstrates that it would be possible to develop specific inhibitors of the N- or C-terminal domain of ACE, making it possible to act on substrates involved in functions other than arterial pressure regulation and the hydrosodium metabolism.
ACE is the main, or even exclusive enzyme in the metabolism of plasma AcSDKP. Administering a single dose of captopril in healthy volunteer subjects increases the plasma level of the peptide 6 to 7-fold. A selective inhibitor of the N-terminal domain would make it possible to obtain such a result without modifying, unlike captopril and the other converting enzyme inhibitors used to date, the metabolism of the peptides playing a role in the control of arterial pressure and the hydrosodium metabolism (angiotensin, bradykinin). Therefore, ACE N-terminal domain inhibitors would be of great interest for the protection of haematopoietic strain cells of patients undergoing aggressive chemotherapy or radiotherapy treatment. The inhibitor could be administered before or concomitantly with the anti-cancer treatment.
In addition, it was recently demonstrated, by Volpert et al in J. Clin. Invest. (1996) 98, 671-679 [7] that captopril, which inhibits both active sites of ACE, could experimentally exert a protective, anti-cancer, effect in vitro and in vivo. The mechanism of this protective effect is not known but most probably involves AcSDKP, due to its properties on the entry in the cell cycle of numerous types of cells. Therefore, a selective inhibitor of the N-terminal domain of ACE, potentialising the plasma level of AcSDKP, without modifying the metabolism of vasoactive peptides, could have a beneficial effect.
Most of the strong ACE inhibitors developed to date and illustrated in FIG. 1 are characterised by the presence of a pseudo-dipeptide cell on which a chemical group capable of interacting favourably with the zinc atom located in the active ACE site has been grafted. Indeed, ACE belongs to the zinc metalloprotease group and is characterised by the presence of a zinc atom in both active sites of the enzyme, the zinc atom playing an essential role in catalysis. Numerous studies have demonstrated that the synthesis of pseudo-peptides comprising chemical groups capable of chelating zinc offered access to very powerful zinc metalloprotease inhibitors. The chemical groups capable of interacting with zinc include in the commercial ACE inhibitors, the HS thiol group (captopril), the CHxe2x80x94COO carboxyalkyl group (enalapril) and the PO2xe2x80x94X phosphoryl group where X=NH,O,CH2 (Fosinopril).
The documents FR-A-2 676 059 [8] and EP-A-0 725 075 [9] illustrate peptide derivatives that can be used as zinc protease inhibitors, which comprise phosphinic type groups to interact with zinc. In FR-A-2 676 059, said derivatives are bacterial collagenase inhibitors while in EP-A-0 725 075, they are selective 24-15 zinc endopeptidase inhibitors, which are inactive with respect to other proteases such an angiotensin-converting enzyme.
In this way, to date, there were no selective inhibitors of the N-terminal site of angiotensin-converting enzyme. The present invention specifically relates to new peptide derivatives comprising a phosphinic group, which are selective inhibitors of the N-terminal site of said enzyme.
According to the invention, said new peptide derivatives comprise the amino acid sequence according to the following formula:
-Asp-Phe-xcexa8(PO2CH2)-Ala-Xaaxe2x80x2-xe2x80x83xe2x80x83I
wherein:
xcexa8(PO2CH2) indicates that the peptide bond (CONH) between Phe and Ala has been replaced by the phosphinic bond PO2CH2, and
Xaaxe2x80x2 represents an amino acid residue. In this sequence, the PO2CH2 group is in PO2xe2x88x92 form; therefore, it is associated with a counter-ion such as K+, Na+ or any other pharmacologically acceptable metal. The type of counter-ion is of no significance since, in water, charged groups are dissociated.
According to a particular embodiment of the invention, the peptide derivative comprises only the four amino acids of this sequence and observes the following formula:
R1-Asp-Phe-xcexa8(PO2CH2)-Ala-Xaaxe2x80x2-NH2xe2x80x83xe2x80x83II
wherein:
R1 represents the acetyl or benzyloxycarbonyl group,
xcexa8(PO2CH2) indicates that the peptide bond (CONH) between Phe and Ala has been replaced by the phosphinic bond PO2CH2, and
Xaaxe2x80x2 represents an amino acid residue.
Preferentially, R1 represents the acetyl group.
In formulas I and II given above, the amino acids used for Xaaxe2x80x2 may be natural or non-natural amino acids, or pseudo-amino acids.
The natural amino acids may be selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, norleucine, lysine, methionine, phenylalanine, proline, hydroxyproline, serine, threonine, tryptophan, tyrosine, valine, nitrophenylalanine, homoarginine, thiazolidine and dehydropoline.
A pseudo-amino acid may be defined as an amino acid wherein the amino or carbonyl function has been replaced by another chemical group.
In said formulas, the amino acids Asp, Phe, Ala and Xaaxe2x80x2 may be in L or D form. Therefore, the peptide derivative may be composed of a single isomer or of a mixture of 4 diastereoisomers, due to the presence of two asymmetric centres in the derivative.
In the peptide derivative according to the invention, the amino acid Xaaxe2x80x2 is preferentially selected from the following amino acids: Pro, Ala, Thr, Lys and Leu which has a low activity with respect to the C-terminal site of ACE.
Preferentially, Xaaxe2x80x2 represents Ala since this amino acid reinforces the activity of the peptide derivative for the inhibition of-the N-terminal site of the enzyme while having a low inhibitory activity on the C-terminal site.
The peptide derivatives according to the invention are different from the peptide derivatives described in EP-A-0 725 075 in that they comprise before the pseudo-Phe an Asp residue which makes it possible to render the peptide derivative inactive with respect to the C-terminal site of angiotensin-converting enzyme and thus provide the required selectivity. In addition, the peptide derivatives according to EP-A-0 725 075 were inactive with respect to this enzyme while the derivatives according to the invention are active with respect to the N-terminal part of said enzyme.
The presence of Asp and pseudo-Phe residues enables the peptide derivatives to act on sub-sites S1 and S2 of the enzyme, which is an unusual property for an ACE inhibitor. An even more surprising fact is that said peptide derivative comprises a carboxylate group in the C-terminal position of its structure, while all the ACE inhibitors described to date have systematically comprised a free carboxamide group. In this way, said peptide derivative appears to be very original bother in terms of its chemical structure and its inhibiting activity.
According to the invention, although any Phe and Ala amino acid configuration may be suitable, it is preferable for Phe to have the R configuration. In addition, it is also preferable for the Ala amino acid residue to have the S configuration. In this way, the preferred peptide derivative observes the formula:
Ac-Asp-(R)Phe-xcexa8(PO2CH2)-(s)Ala-Ala-NH2xe2x80x83xe2x80x83III
wherein:
Ac represents the acetyl group, and
xcexa8(PO2CH2) indicates that the peptide bond (CONH) between Phe and Ala has been replaced by the phosphinic bond (PO2CH2).
The peptide derivatives according to the invention may be prepared using conventional processes such as that described in FR-A-2 676 059 or using a solid phase synthesis process such as that described in EP-A-0 725 075 on the basis of the synthon of the formula:
Fmoc-Phexcexa8(PO(Ad)-CH2)AlaOH
where Fmoc represents the (fluorenylmethoxyl)carbonyl group and Ad represents the adamantyl group, and using 2-chloritrityl resin as a solid substrate.
This synthon may be prepared in particular according to the protocol described by Yiotakis et al in J. Org. Chem., 1996, 61, 6601-6605[10].
The invention also relates to a pharmaceutical formulation selectively inhibiting the N-terminal site of human angiotensin-converting enzyme, which comprises a peptide derivative according to formula I, II or III given above.
Said pharmaceutical formulation can particularly be used to protect haematopoietic strain cells of patients undergoing aggressive chemotherapy or radiotherapy treatment, for example cancer treatment.
The invention also relates to the use of a peptide derivative according to formula I, II or III described above to produce a medicinal product selectively inhibiting the N-terminal site of human angiotensin-converting enzyme.
Such a medicinal product may be intended to regulate the proliferation of haematopoietic strain cells of patients undergoing cancer treatment.
The invention""s other characteristics and advantages will be seen more clearly upon reading the following description, which is naturally given as an illustration and is not restrictive, with reference to the appended figures.